Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants

RNA polymerase III (Pol III) as well as Pol II (35S) promoters are able to drive hairpin RNA (hpRNA) expression and induce target gene silencing in plants. siRNAs of 21 nt are the predominant species in a 35S Pol II line, whereas 24- and/or 22-nucleotide (nt) siRNAs are produced by a Pol III line. The 35S line accumulated the loop of the hpRNA, in contrast to full-length hpRNA in the Pol III line. These suggest that Pol II and Pol III-transcribed hpRNAs are processed by different pathways. One Pol III transgene produced only 24-nt siRNAs but silenced the target gene efficiently, indicating that the 24-nt siRNAs can direct mRNA degradation; specific cleavage was confirmed by 59 rapid amplification of cDNA ends (RACE). Both Pol II- and Pol III-directed hpRNA transgenes induced cytosine methylation in the target DNA. The extent of methylation is not correlated with the level of 21-nt siRNAs, suggesting that they are not effective inducers of DNA methylation. The promoter of a U6 transgene was significantly methylated, whereas the promoter of the endogenous U6 gene was almost free of cytosine methylation, suggesting that endogenous sequences are more resistant to de novo DNA methylation than are transgene constructs. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.

The potential role of lycopene for the prevention and therapy of prostate cancer : from molecular mechanisms to clinical evidence

Lycopene is a phytochemical that belongs to a group of pigments known as carotenoids. It is red, lipophilic and naturally occurring in many fruits and vegetables, with tomatoes and tomato-based products containing the highest concentrations of bioavailable lycopene. Several epidemiological studies have linked increased lycopene consumption with decreased prostate cancer risk. These findings are supported by in vitro and in vivo experiments showing that lycopene not only enhances the antioxidant response of prostate cells, but that it is even able to inhibit proliferation, induce apoptosis and decrease the metastatic capacity of prostate cancer cells. However, there is still no clearly proven clinical evidence supporting the use of lycopene in the prevention or treatment of prostate cancer, due to the only limited number of published randomized clinical trials and the varying quality of existing studies. The scope of this article is to discuss the potential impact of lycopene on prostate cancer by giving an overview about its molecular mechanisms and clinical effects. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury whilst recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, whilst maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size and permeability decreased, whilst computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (~45% to ~86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.

Physiological basis of salt stress tolerance in rice expressing the antiapoptotic gene SfIAP

Programmed cell death-associated genes, especially antiapoptosis-related genes have been reported to confer tolerance to a wide range of biotic and abiotic stresses in dicotyledonous plants such as tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.). This is the first time the antiapoptotic gene SfIAP was transformed into a monocotyledonous representative: rice (Oryza sativa L.). Transgenic rice strains expressing SfIAP were generated by the Agrobacterium-mediated transformation method and rice embryogenic calli, and assessed for their ability to confer tolerance to salt stress at both the seedling and reproductive stages using a combination of molecular, agronomical, physiological and biochemical techniques. The results show that plants expressing SfIAP have higher salt tolerance levels in comparison to the wild-type and vector controls. By preventing cell death at the onset of salt stress and maintaining the cell membrane’s integrity, SfIAP transgenic rice plants can retain plant water status, ion homeostasis, photosynthetic efficiency and growth to combat salinity successfully.

Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis : role of fibroblast growth factor receptor-2

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth.

Human breast cancer cell metastasis to long bone and soft organs of nude mice : a quantitative assay

Bone is a common metastatic site in human breast cancer (HBC). Since bone metastasis occurs very rarely from current spontaneous or experimental metastasis models of HBC cells in nude mice, an arterial seeding model involving the direct injection of the cells into the left ventricle has been developed to better understand the mechanisms involved in this process. We present here a sensitive polymerase chain reaction (PCR) method to detect and quantitate bone and soft organ metastasis in nude mice which have been intracardially inoculated with Lac Z transduced HBC cells. Amplification of genomically incorporated Lac Z sequences in MDA-MB-231-BAG HBC cells enables us to specifically detect these cells in mouse organs and bones. We have also created a competitive template to use as an internal standard in the PCR reactions, allowing us to better quantitate levels of HBC metastasis. The results of this PCR detection method correlate well with cell culture detection from alternate long bones from the same mice, and are more sensitive than gross Lac Z staining with X-gal or routine histology. Comparable qualitative results were obtained with PCR and culture in a titration experiment in which mice were inoculated with increasing numbers of cells, but PCR is more quantifiable, less time consuming, and less expensive. This assay can be employed to study the molecular and cellular aspects of bone metastasis, and could easily be used in conjunction with RT-PCR-based analyses of gene products which may be involved with HBC metastasis.

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct

A major obstacle to 3-dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the model's vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.20±3.14% compared with 3.59±2.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.

Optimal Irrigation and N-fertilizer Management for Sustainable Winter Wheat Production in Khorezm, Uzbekistan

The efficiency of the nitrogen (N) application rates 0, 120, 180 and 240 kg N ha−1 in combination with low or medium water levels in the cultivation of winter wheat (Triticum aestivum L.) cv. Kupava was studied for the 2005–2006 and 2006–2007 growing seasons in the Khorezm region of Uzbekistan. The results show an impact of the initial soil Nmin (NO3-N + NH4-N) levels measured at wheat seeding on the N fertilizer rates applied. When the Nmin content in the 0–50 cm soil layer was lower than 10 mg kg−1 during wheat seeding in 2005, the N rate of 180 kg ha−1 was found to be the most effective for achieving high grain yields of high quality. With a higher Nmin content of about 30 mg kg−1 as was the case in the 2006 season, 120 kg N ha−1 was determined as being the technical and economical optimum. The temporal course of N2O emissions of winter wheat cultivation for the two water-level studies shows that emissions were strongly influenced by irrigation and N-fertilization. Extremely high emissions were measured immediately after fertilizer application events that were combined with irrigation events. Given the high impact of N-fertilizer and irrigation-water management on N2O emissions, it can be concluded that present N-management practices should be modified to mitigate emissions of N2O and to achieve higher fertilizer use efficiency.

Responses of Swietenia macrophylla (King) seedlings to different sizes of canopy openings in mixed mahogany plantations

A study was conducted during 1997-99 at 2 sites in Sri Lanka (Rambukkana and Kurunegala) to investigate the responses of Swietenia macrophylla seedlings to wide, moderate and narrow openings of high to low shade conditions in a mature mixed mahogany plantations. Survival, stem growth and shoot phenology of seedlings were recorded monthly. Seedling survival a year after planting showed high mortality under high shaded gap (3-8% photosynthetically active radiation (PAR)). At 51 weeks after planting, final stem height and root collar diameter were highly significant under low shaded gaps. Increased number of shoots and shoot lenghts were observed under low shade (50-78% PAR). Increased flushing was seen in all shade regimes during the rainy period. This study illustrates that low shaded gap openings favour seeding survival, stem and shoot growth, and number of shoots. On the contrary, high shaded gaps reduce the growth of seedlings and therefore may be less attractive to shoot borers.

Perception of prescription medicine sample packs among Australian professional, government, industry, and consumer organizations, based on automated textual analysis of one-on-one interviews

Background Prescription medicine samples provided by pharmaceutical companies are predominantly newer and more expensive products. The range of samples provided to practices may not represent the drugs that the doctors desire to have available. Few studies have used a qualitative design to explore the reasons behind sample use. Objective The aim of this study was to explore the opinions of a variety of Australian key informants about prescription medicine samples, using a qualitative methodology. Methods Twenty-three organizations involved in quality use of medicines in Australia were identified, based on the authors' previous knowledge. Each organization was invited to nominate 1 or 2 representatives to participate in semistructured interviews utilizing seeding questions. Each interview was recorded and transcribed verbatim. Leximancer v2.25 text analysis software (Leximancer Pty Ltd., Jindalee, Queensland, Australia) was used for textual analysis. The top 10 concepts from each analysis group were interrogated back to the original transcript text to determine the main emergent opinions. Results A total of 18 key interviewees representing 16 organizations participated. Samples, patient, doctor, and medicines were the major concepts among general opinions about samples. The concept drug became more frequent and the concept companies appeared when marketing issues were discussed. The Australian Pharmaceutical Benefits Scheme and cost were more prevalent in discussions about alternative sample distribution models, indicating interviewees were cognizant of budgetary implications. Key interviewee opinions added richness to the single-word concepts extracted by Leximancer. Conclusions Participants recognized that prescription medicine samples have an influence on quality use of medicines and play a role in the marketing of medicines. They also believed that alternative distribution systems for samples could provide benefits. The cost of a noncommercial system for distributing samples or starter packs was a concern. These data will be used to design further research investigating alternative models for distribution of samples.

Melt electrospinning writing

This thesis has developed an innovative additive manufacturing technology platform, which combines melt electrospinning with direct writing, allowing the fabrication of a new class of highly-ordered ultrafine fibrous materials. Bioresorbable polymer fibres were printed using a variety of designs, with filament resolutions not demonstrated by established melt-extrusion based direct writing processes, to form novel medical devices. This platform was applied to tissue engineering scaffold design, where structures were prepared in a variety of shapes and forms, characterised and then seeded with cells to investigate their biocompatibility, cell-seeding and proliferation behaviour as well as the ability to guide cell growth and differentiation.

The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues

Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced microtissues fall into common microwells. Herein we describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells; the microwell-mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells. To demonstrate the utility of this platform, we used the microwell-mesh to manufacture hundreds of cartilage microtissues, each formed from 5 × 10(3) bone marrow-derived mesenchymal stem/stromal cells (MSC). The microwell-mesh enabled reliable microtissue retention over 21-day cultures that included multiple full medium exchanges. Cartilage-like matrix formation was more rapid and homogeneous in microtissues than in conventional large diameter control cartilage pellets formed from 2 × 10(5) MSC each. The microwell-mesh platform offers an elegant mechanism to retain microtissues in microwells, and we believe that this improvement will make this platform useful in 3D culture protocols that require multiple medium exchanges, such as those that mimic specific developmental processes or complex sequential drug exposures.

Manufacturing and use of human placenta-derived mesenchymal stromal cells for phase I clinical trials: Establishment and evaluation of a protocol

Background/Aim. Mesenchymal stromal cells (MSCs) have been utilised in many clinical trials as an experimental treatment in numerous clinical settings. Bone marrow remains the traditional source tissue for MSCs but is relatively hard to access in large volumes. Alternatively, MSCs may be derived from other tissues including the placenta and adipose tissue. In an initial study no obvious differences in parameters such as cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability, were detected when we compared human marrow derived- MSCs to human placenta-derived MSCs. The aim of this study was to establish and evaluate a protocol and related processes for preparation placenta-derived MSCs for early phase clinical trials. Methods. A full-term placenta was taken after delivery of the baby as a source of MSCs. Isolation, seeding, incubation, cryopreservation of human placentaderived MSCs and used production release criteria were in accordance with the complex regulatory requirements applicable to Code of Good Manufacturing Practice manufacturing of ex vivo expanded cells. Results. We established and evaluated instructions for MSCs preparation protocol and gave an overview of the three clinical areas application. In the first trial, MSCs were co-transplanted iv to patient receiving an allogeneic cord blood transplant as therapy for treatmentrefractory acute myeloid leukemia. In the second trial, MSCs were administered iv in the treatment of idiopathic pulmonary fibrosis and without serious adverse effects. In the third trial, MSCs were injected directly into the site of tendon damage using ultrasound guidance in the treatment of chronic refractory tendinopathy. Conclusion. Clinical trials using both allogeneic and autologous cells demonstrated MSCs to be safe. A described protocol for human placenta-derived MSCs is appropriate for use in a clinical setting, relatively inexpensive and can be relatively easily adjusted to a different set of regulatory requirements, as applicable to early phase clinical trials.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10-5), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10-4) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10-9). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.